Questo lavoro è stato sostenuto da NIH / NIBIB sovvenzioni R00 EB009105 (per NFS) e P30 EB011317 (per NFS), il NIH / NIBIB formazione concessione T32 EB007509 (a AMW), un Case Western Reserve University Interdisciplinare Alliance Investment Grant (per NFS), e una causa Comprehensive Cancer Center di sovvenzione P30 CA043703 (per NFS). Ringraziamo i ricercatori laboratorio per studenti universitari Steinmetz per le loro mani-sul sostegno: Nadia Ayat, Kevin Chen, Sourav (Sid) Dey, Alice Yang, Sam Alexander, Craig D&#39;Cruz, Stephen Hern, Lauren Randolph, Brian Quindi, e Paul Chariou .

1. VNP (CPMV, BMV, PVX e TMV) Propagazione <ol><li> Impostare la camera di impianto al coperto di controllo per 15 ore del giorno (100% di luce, 25 ° C, 65% di umidità) e 9 ore di notte (0% luce, 22 ° C, 60% di umidità). </li><li> Inoculare le piante in base alla timeline nella Tabella 1. </li></ol><table border="1"><tbody><tr><td> CPMV </td><td> PVX, TMV, e BMV </td></tr><tr><td rowspan="2"> Giorno 0: Stabilimento 3 fagiolo dall&#39;occhio semi / pot. </td><td> Giorno 0: Pianta ~ 30 N. benthamiana semi / pot. Concimare una volta alla settimana con 1 cucchiaio di concime / 5 L di acqua. </td></tr><tr><td> Giorno 14: Re-pot N. benthamiana a 1 pianta / vaso. </td></tr><tr><td> Giorno 10: Infect Foglie primarie con CPMV (5 μg/50 pl / foglia) da inoculazione meccanica con una leggera spolverata di carborundum. </td><td> Giorno 28: Infect tre a five lascia con PVX, TMV, o BMV (5 μg/50 pl / foglia) da inoculazione meccanica con una leggera spolverata di carborundum. </td></tr><tr><td> Foglie Harvest e conservarlo in -80 ° C.: Giorno 20 </td><td> Foglie Harvest e conservarlo in -80 ° C.: giorno 42 </td></tr></tbody></table> Tabella 1. Timeline per la coltivazione, infettando, e la raccolta foglie. Nota: solo propagazione CPMV è dimostrato come esempio. 2. VNP (CPMV, BMV, PVX e TMV) Purificazione Nota: Tutte le operazioni sono eseguite su ghiaccio oppure a 4 ° C. <ol><li> Omogeneizzare 100 g di foglie congelati in un frullatore standard utilizzando due volumi di tampone freddo (vedi Tabella 2). Filtrare 2-3 strati di garza. </li><li> Per PVX, regolare il pH a 6,5 ​​con 1 M HCl. Aggiungere 0,2% (w / v) di acido ascorbico e 0,2% (w / v) di sodio sulfite. </li><li> Centrifugare pianta omogenato grezzo a 5.500 xg per 20 min. Raccogliere il surnatante. </li><li> Per BMV, strato 25 ml di surnatante oltre 5 ml di 10% (w / v) di saccarosio. Centrifugare a 9000 xg per 3 ore e risospendere pellet in soluzione CsCl 38,5% (w / v). Mescolare agitando per 5 ore, poi continuare con il passo 2.12. </li><li> Estrarre materiale vegetale con l&#39;aggiunta di 0,7 volumi di 1:1 (v / v) cloroformio :1-butanolo. Mescolare impasto per 30-60 min. </li><li> Centrifugare soluzione a 5.500 xg per 20 min. Raccogliere la fase acquosa superiore. </li><li> Aggiungere a 0,2 M NaCl e l&#39;8% (w / v) PEG (MW 8000). Per TMV, anche aggiungere 1% (v / v) Triton X-100. Mescolare per almeno 1 ora, quindi lasciate riposare per almeno 1 ora. </li><li> Centrifugare soluzione a 15.000 xg per 15 min. Risospendere il pellet in 10 ml di tampone. Per PVX, aggiungere 0,1% β-mercaptoetanolo e urea a 0,5 M. </li><li> Centrifugare a 8000 xg per 30 minuti e raccogliere il supernatante. </li><li> Ultracentrifuga surnatante a 160.000 xg per 3 ore. Risospendere il pellet in 5 ml di tampone overnight. </li><li> Preparazione di un gradiente di saccarosio 10-40% con volumi pari al 10%, 20%, 30%, e 40% di saccarosio in tampone (più pesante prima). Lasciare che il gradiente di equilibrare notte a temperatura ambiente. </li><li> Ultracentrifuga sedimento risospeso su gradiente di saccarosio a 100.000 xg per 2 ore (24 ore per BMV). </li><li> Raccogliere banda light scattering e dializza contro tampone. </li><li> Caratterizzare le VNPs (sotto) e conservare a 4 ° C. Per la conservazione a lungo termine, conservare a -80 ° C. </li></ol><table border="1"><tbody><tr><td> CPMV e TMV </td><td> 0,1 M tampone fosfato di potassio (pH 7,0) 38,5 mM KH 2 PO 4 61,5 mM K 2 HPO 4 </td></tr><tr><td> PVX </td><td> 0,5 M tampone borato (pH 7,8) 0,5 M di acido borico Aggiustare il pH con NaOH </td></tr><tr><td> BMV </td><td> SAMA tampone (pH 4,5) 250 mM acetato di sodio 10 mM MgCl 2 2 mM β-mercaptoetanolo (aggiungere fresco) </td></tr></tbody></table> Tabella 2. Buffer e le loro ricette per ogni VNP. Nota: solo propagazione CPMV è dimostrato come esempio. 3. VNP (CPMV, BMV, PVX e TMV) Caratterizzazione <ol><li> Effettuare spettroscopia UV / visibile per determinare la concentrazione di VNPs. <ol><li> Misurare l&#39;assorbanza di 2 ml di campione utilizzando uno spettrofotometro NanoDrop. </li><li> Determinare la concentrazione di particelle e coloranti utilizzando la legge di Lambert-Beer (A = εcl, dove A è l&#39;assorbanza, ε è il coefficiente di estinzione, c è la concentrazione, ed L è la lunghezza del percorso). La lunghezza del percorso è di 0,1 cm per la NanoDrop. Le VNP-specifici coefficienti di estinzione sono: CPMV: 8.1 cm-1 mg 1 ml (a 260 nm) PVX: 2.97 cm-1 mg 1 ml (a 260 nm) TMV: 3.0 cm-1 mg 1 ml (a 260 nm) BMV: 5.15 cm-1 mg 1 ml (a 260 nm) </li></ol></li><li> Analizzare le particelle di dimensioni esclusione cromatografia liquida veloce proteine ​​(FPLC). <ol><li> Utilizzando un Superose 6 esclusione dimensionale e la colonna Explorer ÄKTA, carico 50-100 pg di VNPs in 200 pl di tampone fosfato di potassio 0,1 M (pH 7,0). </li><li> Impostare rivelatori a 260 nm (acido nucleico), 280 nm (proteine), e la lunghezza d&#39;onda di eccitazione di eventuali coloranti allegate. </li><li> A una velocità di flusso di 0,5 ml / min per 72 min. </li><li> Il profilo di eluizione e A260: A280 nm indica se la preparazione VNP è puro e se le particelle sono intatti e assemblati. Il seguente A260: 280 rapporti indicano una preparazione pura VNP: CPMV: 1.8 ± 0.1 PVX: 1,2 ± 0,1 TMV:1,1 ± 0,1 BMV: 1.7 ± 0.1 </li></ol></li><li> Eseguire la denaturazione (pre-cast NuPAGE) Bis-Tris poliacrilammide 4-12% elettroforesi su gel di pendenza di analizzare la purezza della preparazione e la coniugazione di proteine ​​di rivestimento individuali. <ol><li> Aggiungere 3 ml di tampone campione 4x LDS a 10 mg di particelle in 9 ml di tampone fosfato di potassio. Aggiungere un ulteriore 1 ml di tampone campione LDS 4x e 3 ml di β-mercaptoetanolo per BMV di ridurre l&#39;elevato numero di legami disolfuro. </li><li> Incubare in blocco termico per 5 minuti a 100 ° C. </li><li> Caricare i campioni su un gel SDS. </li><li> Eseguire i campioni a 200 V per 1 ora in esecuzione 1x MOPS buffer. </li><li> Documentare il gel sotto la luce UV per visualizzare proteine ​​di rivestimento fluorescenti. </li><li> Per non-proteina fluorescente, macchia con blu Coomassie (0,25% (w / v) Coomassie Brilliant Blue R-250, acido 30% (v / v) di metanolo, 10% (v / v) acetico) per 1 ora. </li><li> Decolorare con 30% metanolo, 10% acido acetico durante la notte. Change la soluzione, se necessario. </li><li> Documentare il gel sotto luce bianca. </li></ol></li><li> Analizzare l&#39;integrità delle particelle mediante microscopia elettronica a trasmissione (TEM). <ol><li> Diluire i campioni di 0,1-1 mg / ml in 20 ml di acqua deionizzata. </li><li> Mettere 20 gocce ul dei campioni su parafilm. </li><li> Coprire gocce con una griglia TEM e lasciate riposare per 2 min. Stoppino dalla soluzione in eccesso sulla griglia con carta da filtro. </li><li> Lavare griglia immissione in una goccia di acqua deionizzata quindi assorbente asciutto. </li><li> Stain griglia mettendo su una goccia 20 ml di 2% (w / v) di acetato di uranile per 2 min. Wick l&#39;eccesso macchia con carta da filtro. </li><li> Lavare rete ancora una volta in acqua. </li><li> Osservare griglia sotto un microscopio elettronico a trasmissione. </li></ol></li></ol> 4. Coniugazione chimica del VNPs con PEG e fluorofori, purificazione e caratterizzazione <ol><li> Per i calcoli per le reazioni di sotto, la massa molare dei VNPs sono: CPMV: 5.6x 10 6 g / mol PVX: 35 x 10 6 g / mol TMV: 41 x 10 6 g / mol BMV: 4,6 x 10 6 g / mol </li><li> Coniugare coloranti e PEG per lisine superficiali di CPMV e PVX usando un one-step N-idrossi succinimmide reazione di accoppiamento: Aggiungi 2500 equivalenti molari (tutti gli eccessi molari consultare eccesso molare per VNP) di Alexa Fluor 647 estere succinimidil e 4.500 equivalenti di NHS- PEG (MW 5000) disciolto in DMSO ad CPMV in 0,1 M tampone fosfato di potassio. Quando si lavora con PVX, aggiungere un eccesso molare di 10000 NHS-colorante e NHS-PEG. Regolare i volumi di tampone e DMSO tale che la concentrazione finale di CPMV e PVX è di 2 mg / ml di DMSO e il contenuto è 10% del volume totale di reazione. Incubare la miscela di reazione per una notte a temperatura ambiente al riparo dalla luce. CPMV e PVX avere 300 e 1270 lisine indirizzabili, rispettivamente. (Si rimanda ai seguenti riferimenti per ulteriori letturemodificazione chimica di CPMV e PVX: 13-15). </li><li> Coloranti Coniugato e PEG per tirosine di TMV con il raccordo di diazonio seguito da rame (I)-catalizzata azide-alchino cicloaddizione. <ol><li> Preparare sale di diazonio (alchini) mescolando 400 pl di 0,3 M p-toluensolfonico monoidrato, 25 pl di nitrito di sodio 3,0 M e 75 ml di 0,68 M distillata 3-ethynylaniline disciolto in acetonitrile a 4 ° C per 1 ora. </li><li> Aggiungere 3,3 ml di tampone borato, pH 8,8, contenente 100 mM NaCl a 1,25 ml di TMV (20 mg / ml di soluzione concentrata). </li><li> Reagiscono con il TMV 450 microlitri della sale di diazonio (alchini) soluzione in un bagno di ghiaccio per 3 ore per aggiungere una legatura alchino maniglia a TMV mediante accoppiamento di diazonio. La soluzione si trasformerà in un colore marrone chiaro. TMV ha 2140 tirosine disponibili per coniugazione. </li><li> Purificare il prodotto finale utilizzando un cuscino di saccarosio come descritto nel passaggio 4,4. </li><li> Collegare azide-funzionale Alexa Fluor 647 e PEG-azide (MW 5.000) using di rame (I)-catalizzata azide-alchino cicloaddizione (CuAAC). Aggiungere 2 equivalenti di coloranti e PEG-azide per proteina di rivestimento e incubare con 1 mM CuSO 4, 2 mM AMG, e 2 mM ascorbato di sodio a temperatura ambiente per 15 min. Regolare il volume di accumulo tale che la concentrazione di reazione finale di TMV è di 2 mg / ml. (Si rimanda ai seguenti riferimenti per ulteriori approfondimenti sulla modificazione chimica di TMV: 16,17). </li></ol></li><li> Coniugare tinture per lisine e PEG per cisteine ​​di cisteina mutante BMV (cBMV): <ol><li> Aggiungere 2.000 equivalenti molari di Oregon verde 488 succinimmidil estere disciolti in DMSO al cBMV in 0,1 M TNKM tampone (50 mM Tris base, 50 mM NaCl, 10 mM KCl, 5 mM MgCl 2, pH 7,4). Regolare i volumi di tampone e DMSO tale che la concentrazione finale di BMV è 1 mg / ml di DMSO e il contenuto è 10% del volume totale di reazione. Incubare la miscela di reazione per una notte a 4 ° C al riparo dalla luce. </li><li> Particelle Purificare con centrifugafiltri fugati, come descritto al punto 4.4. </li><li> Aggiungere 2.000 eccesso molare di PEG-maleimmide (MW 2000) utilizzando le stesse condizioni di reazione come prima e incubare la miscela di reazione per 2 ore a 4 ° C. cBMV dispone di 180 lisine reattivi e cisteine. (Si rimanda ai seguenti riferimenti per ulteriori letture sulla modificazione chimica di BMV: 18). </li></ol></li><li> Purificazione: Passare la soluzione attraverso un cuscino di saccarosio al 40% (w / v) a 160.000 xg per 2,5 ore. Nuovamente sciogliere il precipitato in un tampone. In alternativa, dializza contro tampone appropriato con 10 kDa di cut-off filtri rotativi. </li><li> Caratterizzazione: VNPs pegilato e marcato in fluorescenza vengono analizzate utilizzando i metodi sopra descritti: UV / visibile spettroscopia, elettroforesi su gel SDS, FPLC e TEM (non mostrato, tuttavia, fare riferimento alle figure 6 e 7). </li></ol> 5. Targeting tumorale e imaging utilizzando un modello Xenograft mouse<ol><li> Cultura HT-29 cellule umane di cancro al colon in mezzo RPMI supplementato con 5% FBS, 1% di penicillina-streptomicina, e 1% di L-glutammina a 37 ° C in 5% CO 2 con 175 centimetri due fiasche di coltura cellulare. </li><li> Lavare le cellule due volte con PBS sterile e raccolto mediante incubazione con 5 ml di tripsina-EDTA a 37 ° C per 5 min. Inattivare la tripsina con 5 ml di terreno RPMI. Raccogliere le cellule per centrifugazione a 500 xg per 5 min. a 4 ° C e risospendere in fresco RPMI a 5x10 6 cells/50 medie microlitri (determinare il numero totale delle cellule con Trypan Blue e un emocitometro). Mescolare con un volume uguale di matrigel prima dell&#39;iniezione (mantenere tutte le soluzioni e reagenti sterile). </li><li> Procurarsi sei settimana fa NCR nu / nu topi e mantenerli su una dieta di erba medica gratuita per 2 settimane. [Nota:. Tutte le procedure di polizia deve essere approvato IACUC] indurre xenotrapianti tumorali mediante iniezione sottocutanea di 5x10 6 cells/100 pl / tumore nei fianchi (2 tumori / mouse) utilizzando un 18 1/2 calibro sterileago. Monitorare gli animali regolarmente. Misurare la dimensione del tumore, utilizzando pinze e lasciare i tumori di crescere fino a un volume medio di 20 mm 3 (entro i prossimi 12 giorni). Assegnare i topi a due gruppi diversi in modo casuale: PBS e VNP (n = 3 animali / gruppo / ora punto). Utilizzando una siringa da 28 ml uno scartamento insulina, somministrare per iniezione endovenosa vena della coda 100 pl di PBS sterile o 10 mg / kg formulazione VNP. </li></ol> Nota: esperimenti di coltura dei tessuti e gli studi con animali vivi non sarà dimostrato. Hands-on dimostrazione sarà limitata alla lavorazione dei tessuti e acquisizione dati. Per un riferimento sul modello HT-29 xenograft tumorale, si rimanda il lettore al rif. 19 Tre tecniche sono utilizzate per valutare tumore homing di VNPs: <ol start="4"><li> Imaging di fluorescenza utilizzando Maestro Imaging System: topi Sacrifice a tempi diversi (2, 24, e 72 ore) con emissioni di CO 2gas. Staccare gli animali e tutti gli organi principali accise (cervello, cuore, polmoni, milza, reni e fegato), insieme con i tumori sui fianchi, posizionare i tessuti su parafilm, e analizzare con strumenti di imaging di fluorescenza con eccitazione giallo e filtri di emissione (800 ms esposizione) per rilevare la presenza di segnali fluorescenti nei tessuti (derivato da A647 etichetta coniugato ai VNPs). Salvare le immagini e analizzare le intensità di fluorescenza utilizzando ImageJ 1.44o software ( <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a> ). Confrontare il modello della captazione delle VNPs nei tumori con altri tessuti importanti con il tempo. </li><li> Dopo l&#39;imaging, tagliare ogni tessuto a metà e incorporare una metà in OCT per l&#39;analisi crio-sezionamento e confocale. Raccogliere l&#39;altra metà in pre-pesati crio-fiale e immediatamente congelare utilizzando liquido N 2. Conservare a -80 ° C fino al momento per l&#39;ulteriore elaborazione. </li><li> Fluorescenza quantificazione: Recordone tessuti pesi. Scongelare tessuti congelati a temperatura ambiente e metterli in separati provette Falcon da 50 ml contenenti 1 ml di PBS. Utilizzo di un omogeneizzatore palmare tessuto, omogeneizzare i tessuti per 2-3 min in PBS poi trasferire l&#39;omogeneizzato ai tubi microcentrifuga. Centrifugare omogenati per 10 minuti a 13.000 xg per rimuovere tessuto non omogeneizzato. </li><li> Pipettare 100 pl del supernatante dai tessuti di ogni gruppo (formulazioni PBS e VNP / punti temporali) in una piastra a 384 UV ben nero. Valutare l&#39;intensità di fluorescenza (Ex / Em lunghezze d&#39;onda 600/665) utilizzando un lettore di piastre. Normalizzare i valori ottenuti fluorescenti dai pesi dei tessuti. </li><li> Immunoistochimica: Preparare crio-microtomo sezioni (10 micron) e conservare a -20 ° C. Stain sezioni di tessuto per nuclei cellulari (DAPI) e marcatore delle cellule endoteliali (coniugati con fluoresceina anti-topo CD31 anticorpo). Effettuare analisi di microscopia confocale per mappare la localizzazione vascolare e intra-tumorale di fluorescenza marcata VNPs. </li></ol> Nota: Questa procedura non sarà dimostrato, dati rappresentativi sono mostrati in Figura 8. Per un riferimento immunoistochimica ed i metodi di colorazione descritti, si rimanda il lettore al rif. 19

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+of+the+tobacco+mosaic+virus.">Dual-surface modification of the tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Yildiz, I., Tsvetkova, I., Wen, A. M., Shukla, S., Masarapu, M. H., Dragnea, B., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+Brome+mosaic+virus+for+biomedical+applications.">Engineering of Brome mosaic virus for biomedical applications.</a> RSC Advances. , (2012).</li><li>Brunel, F. M., Lewis, J. D., Destito, G., Steinmetz, N. F., Manchester, M., Stuhlmann, H., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano Lett. 10, 1093-1097 (2010).</li><li>Shukla, S., Ablack, A., Wen, A., Lee, K., Lewis, J., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tumor+homing+and+tissue+penetration+of+the+filamentous+plant+viral+nanoparticle+Potato+virus+X.">Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.</a> Molecular Pharmaceutics. , (2012).</li><li>Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S. P., Wong, S. M., Clinton, G., Ghosh, P., Lin, T., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+conjugation+of+heterologous+proteins+on+the+surface+of+Cowpea+mosaic+virus.">Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.</a> Bioconjug. Chem. 15, 807-813 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo fornisce un approccio per la modifica chimica di VNPs e delle loro applicazioni per imaging in vivo del tumore. Le tecniche di imaging di fluorescenza animale, quantificazione fluorescenza, l&#39;immunoistochimica e presentati in questa sede sono utili per lo studio di biodistribuzione e la valutazione del tumore homing. Queste tecniche forniscono informazioni importanti per quanto riguarda l&#39;accesso delle nanoparticelle al tumore attraverso l&#39;effetto EPR. Combinando i risultati dei va...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>VNP production</td>
		</tr>
		<tr>
			<td>Indoor plant chamber</td>
			<td>Percival Scientific</td>
			<td>E-41L2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>V. unguiculata seeds (California black-eye no. 5)</td>
			<td>Burpee</td>
			<td>51771A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N. benthamiana seeds</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.</td>
		</tr>
		<tr>
			<td>Carborundum</td>
			<td>Fisher</td>
			<td>C192-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pro-mix BX potting soil</td>
			<td>Premier Horticulture</td>
			<td>713400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Jack's Professional 20-10-20 Peat-Lite Fertilizer</td>
			<td>JR Peters</td>
			<td>77860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>50.2 Ti rotor</td>
			<td>Beckman</td>
			<td>337901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW 32 Ti rotor</td>
			<td>Beckman</td>
			<td>369694</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optima L-90K ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SLA-3000 rotor</td>
			<td>Thermo Scientific</td>
			<td>07149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SS-34 rotor</td>
			<td>Thermo Scientific</td>
			<td>28020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sorvall RC-6 Plus centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene bottle</td>
			<td>Beckman</td>
			<td>355607</td>
			<td>For SLA-3000 rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>For SS-34 rotor</td>
		</tr>
		<tr>
			<td>Ultra-Clear tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>For sucrose gradient and SW 32 Ti rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>355618</td>
			<td>For pelleting and 50.2 Ti rotor</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop2000c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PowerEase 500 pre-cast gel system</td>
			<td>Invitrogen</td>
			<td>EI8675EU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL (24 ml) size-exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&Auml;KTA Explorer 100 Chromatograph</td>
			<td>GE Healthcare</td>
			<td>28-4062-66</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra X-12R</td>
			<td>Beckman</td>
			<td>392302</td>
			<td>Benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maestro 2</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>Tissue-Tearor</td>
			<td>Biospec Products</td>
			<td>985370-395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>ZEISS</td>
			<td>Libra 200FE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoView laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Chemicals and Reagents</td>
		</tr>
		<tr>
			<td>3-ethynylaniline</td>
			<td>Sigma Aldrich</td>
			<td>498289-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>384 well black plate</td>
			<td>BD Biosciences</td>
			<td>353285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-12% Bis-Tris NuPAGE SDS gel</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X LDS sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher</td>
			<td>A385-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>271004-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 azide</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ml Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC501096</td>
			<td>10 kDa cut-off</td>
		</tr>
		<tr>
			<td>Aminoguanidine hydrochloride</td>
			<td>Acros Organics</td>
			<td>36891-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>A74-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Fisher</td>
			<td>BP101-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Acros Organics</td>
			<td>42285-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MP Biomedicals</td>
			<td>157574</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>09-801K</td>
			<td>P5 grade</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			<td>BioLegend</td>
			<td>102406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Invitrogen</td>
			<td>16210-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid sodium salt</td>
			<td>Acros Organics</td>
			<td>35268-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>VWR</td>
			<td>48366-277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Invitrogen</td>
			<td>NP0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-2k</td>
			<td>MW 2000</td>
		</tr>
		<tr>
			<td>mPEG-N3</td>
			<td>Nanocs</td>
			<td>PG1-AZ-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>mPEG-NHS</td>
			<td>Nanocs</td>
			<td>PG1-SC-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 succinimidyl ester *6-isomer*</td>
			<td>Invitrogen</td>
			<td>O-6149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-toluenesulfonic acid monohydrate</td>
			<td>Acros Organics</td>
			<td>13902-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher</td>
			<td>BP363-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher</td>
			<td>BP362-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher</td>
			<td>BP333-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Acros Organics</td>
			<td>42435-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Amresco</td>
			<td>0628-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S6-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>Ted Pella</td>
			<td>FCF-400Cu</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>TX1568-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>O3446I-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HT-29 cells</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Invitrogen</td>
			<td>31800-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Scientific</td>
			<td>SV30084.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal Studies</td>
		</tr>
		<tr>
			<td>18% Protein Rodent Diet</td>
			<td>Harlan Teklad</td>
			<td>Teklad Global 2018S</td>
			<td>Alfalfa free diet</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>329410</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AHN3637</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NCR nu/nu mice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>CWRU School 
			of Medicine Athymic Animal and Xenograft Core Facility</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>18 1/2 gauge</td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Andwin Scientific</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral nanoparticles for in vivo tumor imaging

L&#39;uso di nanomateriali ha il potenziale per rivoluzionare scienza dei materiali e della medicina. Attualmente, un certo numero di differenti nanoparticelle sono stati studiati per applicazioni di imaging e terapia. Nanoparticelle virali (VNPs) derivati ​​da piante può essere considerata autoassemblati bionanomaterials con dimensioni e forme definite. Virus di piante in esame in laboratorio Steinmetz sono particelle formate icosaedrici dal virus del mosaico Cowpea (CPMV) e virus del mosaico Brome (BMV), entrambi i quali sono 30 nm di diametro. Stiamo anche sviluppando strutture a forma di bastoncello e filamentose derivati ​​dai virus delle piante seguenti: virus del mosaico del tabacco (TMV), che costituisce aste rigide con dimensioni di 300 nm da 18 nm, e Virus X della patata (PVX), che formano le particelle filamentose 515 nm di lunghezza e 13 nm in larghezza (si rimanda il lettore al refs. 1 e 2 per ulteriori informazioni sulla VNPs). <p class="jove_content"&gt; Dal punto uno scienziato dei materiali di vista, VNPs sono elementi interessanti per diversi motivi: le particelle sono monodisperse, può essere prodotta con facilità su larga scala in planta, sono eccezionalmente stabili e biocompatibili. Inoltre, VNPs sono &quot;programmabili&quot; unità, che possono essere specificamente progettati utilizzando modificazione genetica o metodi chimici bioconjugation 3. La struttura di VNPs è noto risoluzione atomica, e modifiche possono essere effettuate con precisione spaziale a livello atomico 4, un livello di controllo che non può essere raggiunto utilizzando nanomateriali di sintesi con le attuali state-of-the-art. In questo articolo, si descrive la propagazione di CPMV, PVX, TMV, e BMV a Vigna ungiuculata e piante di Nicotiana benthamiana. Protocolli di estrazione e purificazione di ogni VNP sono dati. Metodi per la caratterizzazione dei purificato e VNPs chimicamente etichettati sono descritti. In questo studio, ci concentriamo su chetichettatura emical di VNPs con fluorofori (es. Alexa Fluor 647) e polietilenglicole (PEG). I coloranti facilitare il monitoraggio e la rilevazione dei VNPs 5-10, e PEG riduce immunogenicità delle nanoparticelle proteici migliorando loro farmacocinetica 8,11. Dimostriamo tumore homing di VNPs pegilati utilizzando un modello di topo xenotrapianto del tumore. Una combinazione di imaging di fluorescenza di tessuti ex vivo utilizzando Maestro Imaging System, quantificazione fluorescenza nei tessuti omogeneizzati, e microscopia confocale viene utilizzato per studiare biodistribuzione. VNPs vengono cancellati tramite il sistema reticolo-endoteliale (RES); tumore homing avviene passivamente attraverso la permeabilità maggiore e ritenzione (EPR) effetto 12. La nanotecnologia VNP è un potente plug-and-play della tecnologia per l&#39;immagine e il trattamento di siti di malattia in vivo. Stiamo sviluppando ulteriormente VNPs portare carichi di droga e frazioni di imaging clinicamente rilevanti, così come tessuto-specifici ligandi dibersaglio i recettori molecolari sovraespresse nel cancro e malattie cardiovascolari.

<img alt="Figure 1" src="/files/ftp_upload/4352/4352fig1.jpg" /> 
 Figure 1. Plant virus-infected plants. Vigna unguiculata plants infected with CPMV (A). Nicotiana benthamiana plants infected with PVX (B), TMV (C), and BMV (D). The pictures were taken about 10 days post infection by mechanical inoculation.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4352...

Watch this Scientific Journal Video about Viral Nanoparticles for In vivo Tumor Imaging at JoVE.com

Viral Nanoparticles for In vivo Tumor Imaging

Nanoparticelle vegetali virali (VNPs) sono promettenti piattaforme per le applicazioni in campo biomedico. Qui, descriviamo le modalità di propagazione delle piante VNP, purificazione, caratterizzazione e bioconjugation. Infine, mostriamo l&#39;applicazione di VNPs per homing tumore e immagini utilizzando un modello di xenotrapianto mouse e imaging di fluorescenza.

Nanoparticelle virali per<em&gt; In vivo</em&gt; Imaging Tumore

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

viral-nanoparticles-for-in-vivo-tumor-imaging

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JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

17.1K Views.  Case Western Reserve University. Nanoparticelle vegetali virali (VNPs) sono promettenti piattaforme per le applicazioni in campo biomedico. Qui, descriviamo le modalità di propagazione delle piante VNP, purificazione, caratterizzazione e bioconjugation. Infine, mostriamo l'applicazione di VNPs per homing tumore e immagini utilizzando un modello di xenotrapianto mouse e imaging di fluorescenza.

Video: Viral Nanoparticles for In vivo Tumor Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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Bioingegneria

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Semplici dispositivi microfluidici per<em&gt; In vivo</em&gt; Imaging di<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

Analisi delle Targeted proteina virale nanoparticelle distribuito HER2 + Tumori

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

PLGA nanoparticelle formate da singola o doppia emulsione con Vitamina E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Harmonic Nanoparticelle for Regenerative ricerca

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

Forno a microonde-driven sintesi di ossido di ferro Nanoparticelle per il rilevamento rapido di aterosclerosi

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizzando l'angiogenesi da microscopia multifotonica In Vivo in geneticamente 3D-PLGA/nHAp impalcatura per Calvarial riparazione di difetti ossei critici

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Valutazione iniziale di anticorpo-coniugati modificate con peptidi virali-derivati per accumulo cellulare di aumentare e migliorare il Targeting tumorale

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Microscopia a luce correlativa (CLEM) per il monitoraggio e la proteina virale di Imaging strutture in cellule di Cryo-immobilizzata associate

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...